Protease inhibitors are essential components in the chemotherapy of HIV-1 infection. Despite their success, the long-term efficacy of antiretroviral therapies is continuously hindered by the emergence of viral strains that exhibit resistance to protease inhibitors. The onset of drug resistance is often accelerated by therapy lapses associated with the occurrence of severe side effects in patients undergoing highly active anti-retroviral therapy (HAART). In addition, the viral subtypes prevalent in Africa, where the vast majority of HIV infections take place, are not the same as the one responsible for the infections in America and Europe. Complicating things even further, a different HIV virus, HIV-2, although less prevalent than HIV-1, is also able to cause AIDS. It is clear that successful protease inhibitors would have to maintain appropriate potency against a wide range of target variability. As of today, the FDA has approved nine protease inhibitors for clinical use. While all of them target the active site pocket of the same enzyme, they do so with different potency, different resistance profiles and different selectivity towards unwanted targets. An ideal inhibitor should have extremely high potency against the wild type protease, exhibit low susceptibility to protease mutations associated with drug resistance and not interfere with human targets, thus minimizing side effects. The main goal of this project is to develop precise thermodynamic and structural guidelines to develop such inhibitors. The specific goals of this project are: - Development of thermodynamic and structural rules aimed at achieving extremely high affinity. - Identification of thermodynamic and structural determinants that confer protease inhibitors low susceptibility to mutations and efficacy against different viral subtypes, including HIV-2. - Development of thermodynamic and structural rules aimed at limiting the affinity of protease inhibitors to unwanted targets and hence improving selectivity. The goals will be achieved by a combination of experimental thermodynamic measurements (high sensitivity isothermal titration calorimetry and high sensitivity differential scanning calorimetry), structure determination (x-ray crystallography) and structure-based thermodynamic analysis. PUBLIC HEALTH RELEVANCE: More than 30 million people in the world are infected with HIV/AIDS and more than 2 million die each year. Despite their initial success, antiretroviral therapies are hindered by the emergence of drug resistant viral strains and by the occurrence of severe side effects. The main goal of this project is to develop precise guidelines for the development of antiretrovirals, especially protease inhibitors, characterized by extremely high potency, low susceptibility to drug resistance and minimal side effects.